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Phenotypic and Molecular Characterization of Escherichia coli Isolated from Food Animals for Antimicrobial Resistance 

M. Ranjan1, Rahul Kolhe1,*, Chandrakant Bhong1, Sujata Bhave1, Vijaysinh Lonkar2, Sameer Jadhav3, Prashant Mhase4
1Department of Veterinary Public Health, KNP College of Veterinary Science, Maharashtra Animal and Fishery Sciences University, Shirwal-412 801, Maharashtra, India.
2Department of Poultry Science, KNP College of Veterinary Science, Maharashtra Animal and Fishery Sciences University, Shirwal-412 801, Maharashtra, India.
3Department of Veterinary Biochemistry, KNP College of Veterinary Science, Maharashtra Animal and Fishery Sciences University, Shirwal-412 801, Maharashtra, India.
4Department of Veterinary Microbiology, KNP College of Veterinary Science, Maharashtra Animal and Fishery Sciences University, Shirwal-412 801, Maharashtra, India.

ABSTRACT

Background: The use of antimicrobials in food animals significantly contributes to the development of antimicrobial resistance in nature. This study aimed to determine the prevalence of ESBL-producing E. coli isolated from cattle, buffalo, sheep and goat fecal samples. 

Methods: In this study out of 2025 healthy animals from 10 different farms, fecal samples were randomly collected by rectal swabs from 200 animals (50 each from cattle, buffalo, sheep and goat). E. coli was isolated and further studied for AMR and ESBL production by phenotypic assays and PCR targeting ESBL genes. 

Result: The overall prevalence of E. coli was 81.50% with the highest isolation rate from cattle and goat samples. Out of 163 isolates, 45.39% were ESBL-producers. Genes encoding beta-lactam resistance viz. blaSHV, blaTEM, blaCTX-M and blaOXA were detected in 44.59% ESBL-positive isolates. The blaSHV was present in only cattle isolates, while blaCTX-M was present in E. coli isolated from all animals. The prevalence of beta-lactam genes was found as blaSHV (2.70%), blaTEM (12.16%), blaCTX-M (22.97%) and blaOXA (6.75%). A high degree of resistance to multiple antimicrobials was observed. Maximum susceptibility of E. coli was observed for trimethoprim, imipenem and chloramphenicol. This study demonstrated that food animals could be the source of ESBL-producing multidrug-resistant E. coli. The present findings are significant in the context of AMR monitoring in India.

INTRODUCTION

Although the development of antimicrobial resistance (AMR) in the bacterial community is considered an ancient and natural phenomenon, its emergence and widespread distribution have escalated to a great extent in recent the past (Laxminarayan et al., 2013). Livestock and pets are considered as a potential source of AMR spread in nature. The role of livestock, poultry and aquaculture in the spread of AMR has been debated for a long (Marshall and Levy, 2011). There is a correlation between the use of antibiotics and the emergence of resistance to pathogens (Allen et al., 2010). Beta-lactam antibiotics are the most widely used agents for the treatment of bacterial infection, therefore, resistance against Extended Spectrum Beta-Lactamase (ESBL) has been recorded globally. Among ESBL-producing Enterobacteriaceae, Klebsiella species and Escherichia coli (E. coli) are the most commonly resistant bacteria causing community-acquired infections (Pormohammad et al., 2019). Research on AMR in the livestock origin bacterial species with special reference to the critical antimicrobials is at a primitive stage. This study was undertaken on the isolation and characterization of E. coli from fecal samples of cattle, buffalo, sheep and goats in terms of AMR, ESBL production and prevalence of ESBL encoding genes.

MATERIALS AND METHODS

Sampling
 
In this study, farms having at least 50 animals of a single species were identified for sampling. Accordingly, 10 farms located at distinct places in the Satara and Pune Districts of Maharashtra state were selected for sampling. From each farm, rectal swabs of apparently healthy animals were randomly collected by the veterinarian. The proportion of samples collected from each farm varies from 5-10%, which depends on the number of animals at the farm. A total of 200 rectal swabs from livestock species (50 each from cattle, buffalo, sheep and goat) were collected. The convenient sampling plan was used to determine the sample size. E. coli was isolated as pure culture by enrichment of fecal swabs in Enterobacteriaceae Enrichment Broth (EEB) for 24 hr at 37°C followed by selective plating on Eosin Methylene Blue (EMB) agar and incubation at 37°C for 24 hr. Isolates were confirmed using biochemical tests viz. catalase, oxidase, indole, methyl red, Voges-Proskauer and citrate utilization tests.
 
Antimicrobial resistance study
 
The antimicrobial susceptibility pattern was studied by modified Kirby–Bauer disk diffusion and the results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2017). Antimicrobial discs were procured from HiMedia Laboratories, Mumbai. Antimicrobial discs used in this study were: ampicillin (10 µg-1), cefazolin (30 µg-1), cefixime (5 µg-1), chloramphenicol (30 µg-1), ciprofloxacin (5 µg-1), gentamicin (10 µg-1), imipenem (10 µg-1), kanamycin (30 µg-1), tetracycline (30 µg-1) and trimethoprim (5 µg-1).
 
ESBL screening
 
ESBL production was confirmed by the disc diffusion synergy test (DDST) as per the recommendations of CLSI. Antimicrobial discs used for initial screening were cefpodoxime (10 µg-1), ceftazidime (30 µg-1), aztreonam (30 µg-1), cefotaxime (30 µg-1), ceftriaxone (30 µg-1). ESBL confirmation was done by using cephalosporin discs viz. cefotaxime (30 µg-1), cefotaxime/clavulanic acid (10 µg-1), ceftazidime (30 µg-1) and ceftazidime/clavulanic acid (10 µg-1). A difference of ≥5 mm in the zone of inhibition between cephalosporin alone and cephalosporin plus clavulanic acid indicates ESBL production (CLSI, 2017).
 
Detection of genes encoding beta-lactamases
 
ESBL-positive E. coli were further screened by multiplex polymerase chain reaction (PCR) designed previously targeting four beta-lactamase genes viz. blaSHV, blaTEM, blaCTX-M and blaOXA (Fang et al., 2008). For PCR targeting beta-lactam genes, bacterial DNA was extracted by the heat lysis method. Multiplex PCR was performed in 25 µl volume containing 12.5 µl 2x PCR master mix,1 µl each forward and reverse primers (10 pmol/µl), 2 µl DNA template and 2.5 µl nuclease-free water to make final the volume. The cycling conditions were set as initial denaturation (95°C/15 min) followed by 30 cycles of denaturation (94°C/30 sec), annealing (62°C/90 sec) and extension (72°C/60 sec). The final extension was set at 72°C for 10 min and held at 4°C. Five µl of amplified product was further separated by electrophoresis in 1.5% agarose gel dissolved in 0.5x TBE stained by ethidium bromide (0.5 mg/mL).

RESULTS AND DISCUSSION

The prevalence of ESBL-positive E. coli strains and ESBL-encoding genes found in this study is depicted in Table 1. Maximum ESBL-producing E. coli strains were isolated from goats (58.69%), followed by sheep (45.94%), buffalo (42.10%) and cattle (33.33%). Altogether, 74 E. coli strains (45.39%) were ESBL producers. Bacteria belonging to the family Enterobacteriaceae are widely studied around the world for antimicrobial resistance. Among livestock species, cattle-origin E. coli has been extensively investigated for ESBL types at a global scale (Schmid et al., 2013; Pehlivanoglu et al., 2016; Aworh et al., 2022). However, studies on ESBL types of E. coli isolated from other food animals viz. buffalo, sheep and goats are limited (Geser et al., 2012). In India, ESBL production was studied by a few investigators in E. coli isolated from cattle feces (Rawat et al., 2018) and raw foods of animal origin (Bhoomika et al., 2016). All 74 isolates were further studied for the presence of ESBL-encoding genes by multiplex PCR. ESBL encoding genes were detected in 33 (44.59%) ESBL-positive E. coli (Fig 1, 2). Prevalence of blaCTX-M (22.97%) was highest among the ESBL genes, followed by blaTEM (12.16%), blaOXA (6.75%) and blaSHV (2.70%). ESBL gene blaSHV was detected only in cattle-origin E. coli. Similarly, blaOXA and blaTEM genes were absent in the goat and cattle isolates, respectively. The blaCTX-M was ubiquitous and present in E. coli isolates from cattle, buffalo, sheep and goats. Beta-lactam gene-specific prevalence in different food animal species was also determined among cattle (blaSHV -22.22%; blaCTX-M - 55.55%; blaOXA- 22.22%); buffalo (blaTEM -42.85%; blaCTX-M -42.85%; blaOXA-14.28%); sheep (blaTEM -28.57%; blaCTX-M -42.85%; blaOXA 28.57%); and goat (blaTEM -40%; blaCTX-M -60%).
 

Table 1: ESBL positivity and ESBL encoding genes in Escherichia coli.


 

Fig 1: Detection of beta lactam genes in E. coli isolated from cattle.


 

Fig 2: Detection of beta lactam genes in E. coli isolated from buffalo.


       
The antimicrobial resistance and susceptibility pattern of 163 isolates recorded during the study is displayed in Table 2. Escherichia coli expressed a high degree of resistance to cefazolin (96.31%) and tetracycline (61.96%), followed by kanamycin (52.76%), ampicillin (46.62%), cefixime and ciprofloxacin (42.94%, each). E. coli isolates were found highly sensitive to trimethoprim (90.18%), chloramphenicol (81.59%), imipenem (74.84%) and gentamicin (58.28%). Escherichia coli isolated from food animals irrespective of species were multi-drug resistant. Almost all the resistant strains exhibited resistance against a minimum of three antimicrobials tested. Greater percentages of isolates were resistant to kanamycin, ampicillin, ciprofloxacin, cefixime, tetracycline and gentamicin irrespective of the livestock species of its origin. In the present study, the majority of the ESBL-positive isolates were resistant to ampicillin, cefazolin, cefixime and tetracycline. Animal-origin ESBL-producing E. coli seems to be resistant to multiple antimicrobials (Islam et al., 2016; Seni et al., 2016). The present finding is also in agreement with previous researchers from India on the AMR pattern of E. coli isolated from food animals (Mahanti et al., 2013).
 

Table 2: Antimicrobial sensitivity and resistance pattern of Escherichia coli.


       
Extensive studies were not conducted earlier in India on ESBL characterization of E. coli isolated from buffalo, sheep and goats. E. coli is an inhabitant of the gastrointestinal tract of all vertebrates and thus its presence in the fecal matter is obvious, however, detection of ESBL-positive strains is alarming. Present findings are in agreement with a study from Bavaria, Germany wherein, the prevalence of ESBL-producing E. coli in dairy cows and beef cattle was recorded to the tune of 32.8% (Schmid et al., 2013). Comparatively less prevalence of ESBL-positive E. coli from fecal samples of healthy cattle (13.7%) and sheep (8.6%) was recorded in Switzerland (Geser et al., 2012). ESBL-carrying E. coli were isolated from 12.5% rectal samples of beef cattle from Japan which is far lesser than our findings (Hiroi et al., 2012). Comparatively high prevalence of blaCTX-M and less prevalence of blaSHV were recorded as compared to the findings from India (Kar et al., 2015). A study from China showed that food animals are the major reservoirs of multidrug-resistant E. coli (Ho et al., 2011). The global spread and high prevalence of blaCTX-M type ESBL in E. coli is a matter of Veterinary Public Health concern. The genetic basis for the global dissemination of blaCTX-M is poorly understood. Molecular studies have shown a strong association of the blaCTX-M gene with conjugative plasmid and successful bacterial clones (Tamang et al., 2013).
       
In support of the findings on the buffalo-origin E. coli, a study from Iran revealed the prevalence of blaCTX-M (44.8%), blaTEM (35.2%) and absence of blaSHV in the E. coli isolated from fecal samples of water buffalo (Hoseini et al., 2014). Buffalo-origin E. coli of our study also showed an almost similar pattern and blaSHV was not detected. Diversity in the distribution of ESBL genes was detected in the present study, blaCTX-M and blaTEM were the predominant genes. Reports on ESBL characterization of E. coli from fecal samples of sheep and goats are scarce. In our study, more than 45% of sheep and goat isolates were ESBL- positive E. coli. Similarly, beta-lactam genes viz. blaTEM, blaCTX-M and blaOXA were detected in sheep isolates. Goat isolates were only positive for blaTEM and blaCTX-M. The incidence of ESBL-producing E. coli in small ruminants has been poorly studied in most parts of the world (Mandujano et al., 2023). Findings from Turkey revealed blaCTX-M and blaSHV -positive E. coli in the fecal samples of sheep (Pehlivanoglu et al., 2016).
       
Geographical variation persists in the ESBL types and their variants, therefore, the present study is significant in the context of determining the prevalence of ESBL-producing E. coli in livestock species. Due to its ubiquitous nature and ecological niche, E. coli is used as a sentinel species for studying emerging antimicrobial resistance trends. Some of the studies have also indicated that animals might be responsible for the transfer of ESBL-producing bacteria and the ESBL-encoding genes to humans via contact or animal-origin foods (Ewers et al., 2012). These properties make E. coli a potential strain in the transmission of AMR at the animal-human interface. The study of animal-to-human transmission of antibiotic resistance thus requires a greater understanding of genetic interaction and spread that occur in the larger arena of commensal and environmental bacteria. For better management of the emergence and dispersal of ESBL type of resistance, a complete analysis of abundance, diversity and dissemination of resistance genes in pathogenic and commensal E. coli strains has been suggested (Bajaj et al., 2016).

CONCLUSION

Our study observed that cattle, buffalo, sheep and goats harbor ESBL-producing E. coli. The present study reported that ESBL-positive E. coli were multidrug-resistant strains and blaCTX-M and blaTEM are predominant beta-lactam genes present in them. Significant links between the use of antibiotics in food-producing animals and the development of resistance in indicator bacteria need to be elucidated.

CONFLICT OF INTEREST

All the authors declare that they have no conflict of interest.

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